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Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud.

Adamo JE, Moskow JJ, Gladfelter AS, Viterbo D, Lew DJ, Brennwald PJ - J. Cell Biol. (2001)

Bottom Line: The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells.Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis.Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

ABSTRACT
The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.

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Snc1, Bgl2, and Gas1 are all associated with vesicles that accumulate in cdc42-6 cells. Vesicle gradients of 20 to 40% sorbitol were prepared for each of CDC42, cdc42-6, and sec6-4. Normalized volumes of lysed cells (shifted for 1 h to 37°C for sec6-4 and 33°C for CDC42 and cdc42-6) for each strain were layered onto a gradient before a 1.5 h, 71,000 g spin. The gradients were then collected in 16 fractions and these fractions were subjected to SDS-PAGE analysis and blotted with the indicated antibodies.
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fig4: Snc1, Bgl2, and Gas1 are all associated with vesicles that accumulate in cdc42-6 cells. Vesicle gradients of 20 to 40% sorbitol were prepared for each of CDC42, cdc42-6, and sec6-4. Normalized volumes of lysed cells (shifted for 1 h to 37°C for sec6-4 and 33°C for CDC42 and cdc42-6) for each strain were layered onto a gradient before a 1.5 h, 71,000 g spin. The gradients were then collected in 16 fractions and these fractions were subjected to SDS-PAGE analysis and blotted with the indicated antibodies.

Mentions: Bgl2 does not exhibit a detectable change in electrophoretic mobility as it moves through the secretory pathway, presumably because of a limited level of outer chain mannose addition to the single glycosylation site (Mrsa et al., 1993). This precluded a simple gel mobility analysis of the step in the secretory pathway at which Bgl2 traffic was blocked. However, based on the genetic data described above, we suspected that Bgl2 would be retained in post-Golgi secretory vesicles. Mutations in all of the known factors involved in post-Golgi transport result in the accumulation of 80–100-nm vesicles that contain the v-SNAREs Snc1/2. We made use of velocity gradients to separate the cell's membrane components based on their size. Post-Golgi vesicles are quite uniform in size and run as a discrete peak when analyzed on these gradients. Cells were grown for 1 h at the restrictive temperature (37°C for sec6-4 and 33°C for CDC42 and cdc42-6), lysed, and spun through the sorbitol velocity gradient. Fractions were collected and subjected to SDS-PAGE and immunoblot analysis. As expected, gradients from the control sec6-4 mutant displayed a peak of vesicles containing the v-SNARE Snc1/2 and the Bgl2 cargo (Fig. 4). The accumulated Bgl2 in the cdc42-6 mutant was present in a peak of identical size, which also contained Snc1/2. As expected, these peaks of vesicles were absent in wild-type cells where post-Golgi vesicles are much more rare because of their rapid fusion with the plasma membrane. Therefore, the defect in secretion of Bgl2 found in the cdc42-6 mutant is due to a defect at the level of docking and/or fusion of post-Golgi secretory vesicles with the plasma membrane.


Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud.

Adamo JE, Moskow JJ, Gladfelter AS, Viterbo D, Lew DJ, Brennwald PJ - J. Cell Biol. (2001)

Snc1, Bgl2, and Gas1 are all associated with vesicles that accumulate in cdc42-6 cells. Vesicle gradients of 20 to 40% sorbitol were prepared for each of CDC42, cdc42-6, and sec6-4. Normalized volumes of lysed cells (shifted for 1 h to 37°C for sec6-4 and 33°C for CDC42 and cdc42-6) for each strain were layered onto a gradient before a 1.5 h, 71,000 g spin. The gradients were then collected in 16 fractions and these fractions were subjected to SDS-PAGE analysis and blotted with the indicated antibodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198861&req=5

fig4: Snc1, Bgl2, and Gas1 are all associated with vesicles that accumulate in cdc42-6 cells. Vesicle gradients of 20 to 40% sorbitol were prepared for each of CDC42, cdc42-6, and sec6-4. Normalized volumes of lysed cells (shifted for 1 h to 37°C for sec6-4 and 33°C for CDC42 and cdc42-6) for each strain were layered onto a gradient before a 1.5 h, 71,000 g spin. The gradients were then collected in 16 fractions and these fractions were subjected to SDS-PAGE analysis and blotted with the indicated antibodies.
Mentions: Bgl2 does not exhibit a detectable change in electrophoretic mobility as it moves through the secretory pathway, presumably because of a limited level of outer chain mannose addition to the single glycosylation site (Mrsa et al., 1993). This precluded a simple gel mobility analysis of the step in the secretory pathway at which Bgl2 traffic was blocked. However, based on the genetic data described above, we suspected that Bgl2 would be retained in post-Golgi secretory vesicles. Mutations in all of the known factors involved in post-Golgi transport result in the accumulation of 80–100-nm vesicles that contain the v-SNAREs Snc1/2. We made use of velocity gradients to separate the cell's membrane components based on their size. Post-Golgi vesicles are quite uniform in size and run as a discrete peak when analyzed on these gradients. Cells were grown for 1 h at the restrictive temperature (37°C for sec6-4 and 33°C for CDC42 and cdc42-6), lysed, and spun through the sorbitol velocity gradient. Fractions were collected and subjected to SDS-PAGE and immunoblot analysis. As expected, gradients from the control sec6-4 mutant displayed a peak of vesicles containing the v-SNARE Snc1/2 and the Bgl2 cargo (Fig. 4). The accumulated Bgl2 in the cdc42-6 mutant was present in a peak of identical size, which also contained Snc1/2. As expected, these peaks of vesicles were absent in wild-type cells where post-Golgi vesicles are much more rare because of their rapid fusion with the plasma membrane. Therefore, the defect in secretion of Bgl2 found in the cdc42-6 mutant is due to a defect at the level of docking and/or fusion of post-Golgi secretory vesicles with the plasma membrane.

Bottom Line: The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells.Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis.Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

ABSTRACT
The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.

Show MeSH
Related in: MedlinePlus